Juvenile Myelomonocytic Leukemia (JMML) is a lethal myeloproliferative disorder (MPD) of children, characterized by unregulated proliferation of myelomonocytic cells and hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Most patients show hyperactivation of the Ras signaling pathway, including activating mutations in PTPN11, which encodes the protein tyrosine phosphatase, Shp2. It has been shown that, in response to GM-CSF stimulation, gain-of-function mutations in Shp2 result in elevated levels of phospho-Akt, a downstream effector of the phosphoinositol-3-kinase (PI3K) pathway. The lipid kinase PI3K is a heterodimer consisting of one of two regulatory subunits, p851 or p852, and one of three catalytic subunits, p1101, p1102, or p1104, whose action is antagonized by the lipid phosphatase PTEN. Previously, we demonstrated that genetic interruption of p851 results in partial correction of mutant Shp2-induced hyperproliferation in vitro. Accordingly, we hypothesize that gain-of-function mutant Shp2-mediated hyperactivation of the PI3K pathway contributes to the etiology of JMML. To explore this hypothesis, we propose the following three aims: (1) We will use murine hematopoietic cells lacking expression of both PI3K regulatory subunits p851 and p852, with activating PTPN11 mutants to explore the hypothesis that both p85 subunits contribute to gain-of-function mutant Shp2-mediated GM-CSF hypersensitivity. These studies will include (A) in vitro biochemical and functional analysis, and (B) in vivo transplant studies to examine the development of MPD. (2) We will cross conditional Shp2 D61Y gain-of-function knockin mice with conditional PTEN knockout mice to assess if PTEN deficiency will (A) accelerate and exacerbate development of activating PTPN11-induced MPD in vivo, and (B) result in increased Shp2 D61Y-induced hyperproliferation and Akt phosphorylation in vitro. (3) Finally, to explore the hypothesis that reduced nuclear PTEN contributes to gain-of-function mutant Shp2-induced PI3K hyperactivation, we will (A) co-transduce conditional PTEN knockout bone marrow cells with gain-of-function mutant Shp2 E76K and WT PTEN or mutant PTEN K289E, which abrogates monoubiquitination-mediated nuclear translocation, in order to investigate if inhibition of PTEN nuclear localization exacerbates Shp2 E76K-induced hyperproliferation; and (B) co-transduce conditional PTEN knockout bone marrow cells with WT Shp2 or Shp2 E76K and PTEN-NLSSV-40, which forces nuclear localization of PTEN, to investigate whether gain-of-function mutant Shp2 E76K causes decreased nuclear localization of PTEN by promoting its egress from the nucleus. We expect that the results of these studies will enhance our understanding of the role of PI3K signaling in the pathophysiology of activating PTPN11-induced JMML, potentially providing novel molecular targets for this disease.